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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jun 2006 - 01 Feb 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Exposure from gestational day 6-17 only.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
yes
Remarks:
Exposure gestational days 6-17 only
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
85883-73-4

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 70 days
- Weight at study initiation: 222-295 g (females)
- Housing: upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board; the rats were paired for mating in the home cage of the male; following positive evidence of mating, the females were returned to individual suspended wire-mesh cages.
- Diet: basal diet Certified Rodent LabDiet 5002 (PMI Nutrition International, LLC), ad libitum
- Water: municipal water (reverse osmosis-purified, on-site), ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The daily aliquots were prepared weekly, stored refrigerated under nitrogen when not in use, and allowed to stand at room temperature for at least 1 h prior to administration.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test article was administered neat. Therefore, no analyses were conducted by the Analytical Chemistry Department of the testing laboratory (WIL Research Laboratories, LLC).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug/sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Day 6 -17 of gestation
Frequency of treatment:
daily, 7days/week
Duration of test:
Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
other: Yes, administration of water at 2.65 mL/kg (highest dose volume).
Details on study design:
- Dose selection rationale: dosage levels were selected based on the results of previous studies and were selected by the sponsor following consultation with the WIL study director. A high-dose of 2500 mg/kg/day was chosen as it meets or exceeds the amount expected to be used for clinical purposes.
- The dosage volumes used to obtain the desired dosage levels were based on the specific gravity of the test article at approximately 20ºC (0.942 g/mL); 0.53, 1.59 and 2.65 mL of neat test substance were administered.
- Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: signs of toxicity, moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the time of dose administration and approximately 1 h following dose administration from gestation Days 0 through 20

BODY WEIGHT: Yes
- Time schedule for examinations: gestation Days 0, 6-18 (daily) and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal calculated as g/animal/day and g/kg/day for the corresponding body weight change intervals
- Time schedule for examinations: on gestation Days 0, 6-18 (daily) and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: placenta, uterus and ovaries

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group. Each mean was presented with standard deviation (S.D.) and the number if animals (N) used to calculated the mean. In addition, percent difference from control was presented for body weights. Data obtained from nongravid animals were excluded from statistical analyses. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ± 1 in the last significant figure. Where applicable, the litter was used as the experiment unit.
Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p < 0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal and combined) and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p < 0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test article-treated groups to the control group.
Indices:
Intrauterine data were summarized using 2 methods of calculation:
1) Group Mean Litter Basis: Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group/No. Gravid Females/Group

2.) Proportional Litter Basis:
Summation Per Group (%) = ∑ Postimplantation Loss/Litter (%)/No. Litters/Group
Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter/No. Implantation Sites/Litter) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = ∑ Viable Fetuses Affected/Litter (%)/No. Litters/Group
Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter/No. Viable Fetuses/Litter) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All females in the control, 500, 1500 and 2500 mg/kg bw/day groups survived to the scheduled necropsy on gestation Day 20. In the 1500 and 2500 mg/kg bw/day groups, test article-related salivation was noted for 8 and 6 females, respectively (1-2 occasions each), immediately prior to dose administration on gestation Days 15-17. In addition, salivation-related findings (clear material on various body surfaces) were noted for 2 and 3 females in these same respective groups (1-2 occasions each) approximately 1 h following dose administration, primarily on gestation Days 6-7 and 14-15. Other findings noted in the treated groups including hair loss, scabbing and red material on various body surfaces, as well as rales and soft stool, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mean maternal body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 500, 1500 and 2500 mg/kg bw/day groups were unaffected by test article administration. Differences from the control group were slight and not statistically significant.
A slightly lower food consumption observed in the 2500 mg/kg/day group during gestation Days 6-9, 9-12, 12-18 and when the entire treatment period was evaluated. This finding was not considered adverse based on the lack of an effect on mean body weights.
No other treatment related findings were noted.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 2 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 2 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Postimplantation loss, live litter size, mean fetal body weights, fetal sex ratios, mean numbers of corpora lutea and implantation sites and the mean litter proportions of preimplantation loss were similar across all groups.
The numbers of fetuses (litters) available for morphological evaluation were 336(23), 361(24), 358(25) and 357(24) in the control, 500, 1500 and 2500 mg/kg bw/day groups, respectively. Malformations were observed in 1(1), 2(2), 1(1) and 0(0) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. A malformation was also noted for 1 late resorption in the 500 mg/kg bw/day group.
Fetus no. 38738-01 in the 1500 mg/kg bw/day group had localised fetal edema (head) and fetus no. 38714-12 in the 500 mg/kg bw/day group had microphthalmia (unilateral); skeletally, the left orbit appeared smaller than normal. Vertebral agenesis (all vertebrae posterior to lumbar vertebra no. 2 absent) was noted for a single fetus (no. 38742-05) in the 500 mg/kg bw/day group, and 1 late resorption in the 500 mg/kg/day group had fetal edema. One fetus (no. 38773-13) in the control group had anophthalmia. No external developmental variations were noted for any fetuses in this study.
No visceral malformations were noted for fetuses in this study.
There were no skeletal malformations noted for fetuses at any dosage level in this study.
Fetal malformations and developmental variations, when observed in the test article-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner and/or were within the historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test article.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
embryotoxic
Effect level:
>= 2 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
>= 2 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Based on the lack of adverse findings at 500, 1500 and 2500 mg/kg bw/day, a dosage level of 2500 mg/kg bw/day, the highest dosage level tested, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo/fetal developmental toxicity when the test substance was administered orally by gavage to pregnant Crl:CD(SD) rats from gestation Days 6-17.

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on intrauterine development.